Lignocellulose Biodegradation and Applications in Biotechnology

Chapter 1

Lignocellulose Biodegradation and Applications in Biotechnology

Downloaded by UNIV OF GUELPH LIBRARY on September 12, 2012 | http://pubs.acs.org Publication Date: July 29, 2004 | doi: 10.1021/bk-2004-0889.ch001

Badal C. Saha Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604

Lignocellulosic biomass such as agricultural and forestry residues and herbaceous energy crops can serve as low cost feedstocks for production of fuel ethanol and other value-added commodity chemicals. However, development of efficient pretreatment and cost-effective enzymatic conversion of any lignocellulosic biomass to fermentable sugars is a key issue. In this overview chapter, various pretreatment options (dilute acid, steam explosion, alkaline peroxide) and enzymes (mainly cellulases and hemicellulases) involved in lignocellulose degradation are presented. Mixed sugars generated by lignocellulose biodegradation are fermented to fuel ethanol, xylitol, 2-3-butanediol and other value-added products. Recent advances in the developments on lignocellulose biodegradation and applications in biotechnology are reviewed.

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U.S. government work. Published 2004 American Chemical Society

In Lignocellulose Biodegradation; Saha, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.


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In 2003, about 2.81 billion gallons of ethanol are produced annually in the United States, with approximately 95% derived from fermentation of com starch. With increased attention to clean air and oxygenates for fuels, opportunities exist for rapid expansion of the fuel ethanol industry. Various lignocellulosic biomass such as agricultural residues, wood, municipal solid wastes and wastesfrompulp and paper industry can serve as low cost and abundant feedstocks for production of fuel ethanol or value-added chemicals. It is estimated that approximately 50 billion gallons of ethanol could be producedfromcurrent biomass wastes with the potential to produce up to 350 billion gallons from dedicated energy farms in the USA (/). At present, the degradation of lignocellulosic biomass to fermentable sugars represents significant technical and economic challenges, and its success depends largely on the development of highly efficient and cost-effective enzymes for conversion of pretreated lignocellulosic substrates to fermentable sugars. In this overview chapter, the author reviews the current knowledge on lignocellulose biodegradation and use of lignocellulosic hydrolyzates as feedstocks for developing bio-based products and processes.

Structure and Composition of Lignocellulosic Biomass Lignocellulosic biomass includes various agricultural residues (straws, hulls, stems, stalks), deciduous and coniferous woods, municipal solid wastes (MSW, paper, cardboard, yard trash, wood products), waste from pulp and paper industry and herbaceous energy crops (switchgrass, barmudagrass). The compositions of these materials vary. The major component is cellulose (35-50%), followed by hemicellulose (20-35%) and lignin (10-25%). Proteins, oils and ash make up the remaining fraction of lignocellulosic biomass (/). The structures of these materials are complex with recalcitrant and heterogeneous characteristics and native lignocellulose is resistant to an enzymatic hydrolysis. In the current model of the structure of lignocellulose, cellulose fibers are embedded in a lignin-polysaccharide matrix. Xylan may play a significant role in the structural integrity of cell walls by both covalent and non-covalent associations (2). Cellulose is a linear polymer of D-glucose units linked by 1,4-B-D-glucosidic bonds. Hemicelluloses are heterogeneous polymers of pentoses (xylose, arabinose), hexoses (mannose, glucose, galactose), and sugar acids. Unlike cellulose, hemicelluloses are not chemically homogeneous. Hardwood hemicelluloses contain mostly xylans, whereas softwood hemicelluloses contain mostly glucomannans (5). Xylans of many plant materials are heteropolysaccharides with homopolymeric backbone chains of 1,4-linked P-Dxylopyranose units. Besides xylose, xylans may contain arabinose, glucuronic acid or its 4-O-methyl ether, and acetic, ferulic and p-coumaric acids. The frequency and composition of branches are dependent on the source of xylan (4). The backbone consists of O-acetyl, a-L-arabinofuranosyl, a-1,2-linked glucuronic or 4O-methylglucuronic acid substituents. However, unsubstituted linear xylans have also been isolated from guar seed husk, esparto grass and tobacco stalks (5).



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Xylans can thus be categorized as linear homoxylan, arabinoxylan, glucuronoxylan and glucuronoarabinoxylan. Xylansfromdifferent sources, such as grasses, cereals, softwood and hardwood, differ in composition. Birch wood (Roth) xylan contains 89.3 % xylose, 1% arabinose, 1.4% glucose and 8.3% anhydrouronic acid (6). Rice bran neutral xylan contains 46% xylose, 44.9% arabinose, 6.1% galactose, 1.9% glucose and 1.1% anhydrouronic acid (7). Wheat arabinoxylan contains 65.8% xylose, 33.5% arabinose, 0.1 % mannose, 0.1 % galactose and 0.3% glucose (8). Cora fiber xylan is one of the complex heteroxylans containing P-(l,4)-linked xylose residues (9). It contains 48-54% xylose, 33-35% arabinose, 5-11% galactose and 3-6% glucuronic acid (10). About 80% of the xylan backbone is highly substituted with monomeric side-chains of arabinose or glucuronic acid linked to 0-2 and/or 0-3 of xylose residues and also by oligomeric side chains containing arabinose, xylose and sometimes galactose residues (7 /). The heteroxylans, which are highly cross-linked by diferulic bridges, constitute a network in which the cellulose microfibrils may be imbedded (12). Structural wall proteins might be cross-linked together by isodityrosine bridges and with feruloylated heteroxylans, thus forming an insoluble network (13). Ferulic acid is covalently cross-linked to polysaccharides by ester bonds and to components of lignin mainly by ether bonds (14). In softwood heteroxylans, arabinofuranosyl residues are esterified with /7-coumaric acids and ferulic acids (75). In hardwood xylans, 60-70% of the xylose residues are acetylated (16). The degree of polymerization of hardwood xylans (150-200) is higher than that of softwoods (70-130).

Pretreatment of Lignocellulosic Biomass The pretreatment of any lignocellulosic biomass is crucial before enzymatic hydrolysis. The objective of pretreatment is to decrease the crystallinity of cellulose which enhances the hydrolysis of cellulose by cellulases (17). Various pretreatment options are available to fractionate, solubilize, hydrolyze and separate cellulose, hemicellulose and lignin components (1, 18-20). These include concentrated acid (21), dilute acid (22), S 0 (25), alkali (24, 25), hydrogen peroxide (26), wetoxidation (27), steam explosion (autohydrolysis) (28), ammonia fiber explosion (AFEX) (29), C 0 explosion (30), liquid hot water (31) and organic solvent treatments (32). In each option, the biomass is reduced in size and its physical structure is opened. Some methods of pretreatment of Lignocellulose is given in Table I. The effectiveness of dilute acids to catalyze the hydrolysis of hemicellulose to its sugar components is well known. Two categories of dilute acid pretreatment are used: High temperature (> 160°C) continuous-flow for low solids loading (5-10%, w/w) and low temperature (< 160°C) batch process for high solids loading (10-40%, w/w) (33). Dilute acid pretreatment at high temperature usually hydrolyzes hemicellulose to its sugars (xylose, arabinose and other sugars) that are water 2

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5 Table I. Methods for pretreatment of lignocellulosic biomass

Method

Example

Autohydrolysis

Liquid hot water, steam pressure, steam explosion, supercritical C0 explosion Dilute acid (H S0 ), Concentrated acid (H S0 ) Sodium hydroxide, lime, ammonia, alkaline hydrogen peroxide Methanol, ethanol, butanol, phenol 2

Acid treatment

2

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Alkali treatment Organic solvent with water

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soluble {IS). The residue contains cellulose and often much of the lignin. The lignin can be extracted with solvents such as ethanol, butanol, or formic acid. Alternatively, hydrolysis of cellulose with lignin present produces water-soluble sugars and the insoluble residues that are lignin plus unreacted materials. Torget et al. (34) achieved both high xylan recovery and high simultaneous saccahrification and fermentation (SSF) conversion while applying extremely dilute H S 0 (0.07 wt %) in a counter-current flowthrough configuration. A major problem associated with the dilute acid hydrolysis of lignocellulosic biomass is the poor fermentability of the hydrolyzates. A drawback of the concentrated acid process is the costly recovery of the acid. Steam explosion provides effective fractionation of lignocellulosic components at relatively low costs (35). Optimal solubilization and degradation of hemicellulose are generally achieved by either high temperature and short residence time (270°C, 1 min) or lower temperature and longer residence time (190°C, 10 min) steam explosion (36). The use of S 0 as a catalyst during steam pretreatment results in the enzymatic accessibility of cellulose and enhanced recovery of the hemicellulose derived sugars (37). Steam pretreatment at 200-210°C with the addition of 1% S0 (w/w) was superior to other forms of pretreatment of willow (38). A glucose yield of 95%, based on the glycan available in the raw material, was achieved. Steam explosion can induce hemicellulose degradation to furfural and its derivatives and modification of the lignin-related chemicals under high severity treatment (> 200°C, 3-5 min, 2-3% S0 ) (39). Boussaid et al. (40) recovered around 87% of the original hemicellulose component in the water-soluble stream by steam explosion of Douglas fir softwood under low severity conditions (175°C, 7.5 min, 4.5% S0 ). More than 80% of the recovered hemicellulose was in monomeric form. Enzymatic digestibility of the steam-exploded Douglas-fir wood chips (105°C, 4.5 min, 4.5% S0 ) was significantly improved using an optimized alkaline peroxide treatment (1% H 0 , pH 11.5 and 80°C, 45 min) (41). About 90% of the lignin in the original 2

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wood was solubilized by this procedure, leaving a cellulose-rich residue that was completely hydrolyzed within 48 h, using an enzyme (cellulase) loading of 1 OFPU/g cellulose. Saccharification of 100 g sugarcane bagasse with enzymes after steam explosion with 1% H S0 at 220°C for 30 sec at water to solid ratio of 2:1 yielded 65.1 g sugar (42). A pretreatment method involves steeping of the lignocellulosic biomass (using corn cob as a model feedstock) in dilute NH OH at ambient temperature to remove lignin, acetate and extractives (43). This is followed by dilute acid treatment that readily hydrolyzes the hemicellulose fraction to simple sugars, primarily xylose. The residual cellulose fraction of biomass can then be enzymatically hydrolyzed to glucose. Sugarcane bagasse, corn husk and switchgrass were pretreated with ammonia water to enhance enzymatic hydrolysis (44). Garrote et al. (45) treated Eucalyptus wood substrates with water under selected operational conditions (autohydrolysis reaction) to obtain a liquid phase containing hemicellulose decomposition products (mainly acetylated xylooligosaccharides, xylose and acetic acid). In a further acid catalyzed step (posthydrolysis reaction), xylooligosaccharides were converted into xylose. Wet oxidation method can be used for fractionation of lignocellulosics into solubilized hemicellulose fraction and a solid cellulose fraction susceptible to enzymatic saccharification. Bjerre et al. (46) found that combination of alkali and wet oxidation did not generate furfural and 5-hydroxymethyl furfural (HMF). Klinke et al. (47) characterized the degradation products from alkaline wet oxidation (water, sodium carbonate, oxygen, high temperature and pressure) of wheat straw. ApartfromC 0 and water, carboxylic acids were the main degradation productsfromhemicellulose and lignin. Aromatic aldehyde formation was minimized by the addition of alkali and temperature control. Oxygen delignification of kraft pulp removed up to 67% of the lignin from softwood pulp and improved the rate and yield from, enzymatic hydrolysis by up to 111% and 174%, respectively (48). Palm and Zacchi (49) extracted 12.5 g of hemicellulose oligosaccharides from 100 g of dry spruce using a microwave oven at 200°C for 5 min. Supercritical C 0 explosion was found to be effective for pretreatment of cellulosic materials before enzymatic hydrolysis (50, 51). Zheng et al. (52) compared C 0 explosion with steam and ammonia explosion for pretreatment of sugarcane bagasse and found that C 0 explosion was more cost-effective than ammonia explosion and did not cause the formation of inhibitory compounds that could occur in steam explosion. Phenolic compounds from lignin degradation, furan derivatives (furfural and HMF) from sugar degradation and aliphalic acids (acetic acid, formic acid and levulinic acid) are considered to be fermentation inhibitors generated from pretreated lignocellulosic biomass (53). The formation of these inhibitors depends on the process conditions and the lignocellulosic feedstocks (54). Various methods for detoxification of the hydrolyzates have been developed (55). These include 2

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treatment with ion-exchange resins, charcoal or ligninolytic enzyme laccase, pre-fermentation with the filamentous fungus Trichoderma reesei, removal of non-volatile compounds, extraction with ether or ethyl acetate and treatment with alkali (lime) or sulfite. Treatment with alkali (overliming) has been widely used for detoxification of lignocellulosic hydrolyzates prior to alcohol fermentation. However, overliming is a costly method which also produces low-value byproducts such as gypsum (56). Softwood hydrolyzate, when overlimed with wood ash, improved its fermentability to ethanol which is due to the reduction of the inhibitors such as fiiran and phenolic compounds and to nutrient effects of some inorganic components from the wood ash on the fermentation (57). Persson et al. (58) employed countercurrent flow supercritical fluid extraction to detoxify a dilute acid hydrolyzate of spruce prior to ethanol fermentation with baker's yeast. Weil et al. (59) developed a method for the removal of furfural from biomass hydrolyzate by using a polymeric adsorbent, XAD-4, and desorption of the furfural to regenerate the adsorbent using ethanol. Bjorklund et al. (60) explored the possibility of using lignin residue left after acid hydrolysis of lignocellulosic material for detoxification of spruce dilute acid hydrolyzates prior to fermentation with Saccharomyces cerevisiae. Treatment with the lignin residue removed up to 53% of the phenolic compounds and up to 68% of the furan aldehydes in a spruce dilute acid hydrolyzate. Up to 84% of the lignin-derived compounds can be extracted with organic solvents (ethyl acetate and diethyl ether) from Eucalyptus wood acid hydrolyzate (61). The phenolic compounds extracted by solvents showed antioxidant activity. Each pretreatment method offers distinct advantages and disadvantages. The pretreatment of lignocellulosic biomass is an expensive procedure with respect to cost and energy.

Cellulose Biodégradation Effective hydrolysis of cellulose to glucose requires the cooperative action of three enzymes: endo-1, 4-fl-glucanase (EC 3.2.1.4), exo-1, 4-B-glucanase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21). Cellulolytic enzymes with β-gîucosidase act sequentially and cooperatively to degrade crystalline cellulose to glucose. Endoglucanase acts in a random fashion on the regions of low crystallinity of the cellulosic fiber whereas exoglucanase removes cellobiose (β-1, 4 glucose dimer) units from the non-reducing ends of cellulose chains. Synergism between these two enzymes is attributed to the endo-exo form of cooperativity and has been studied extensively between cellulases in T. reesei in the degradation of cellulose (62). Besides synergism, the adsorption of the cellulases on the insoluble substrates is a necessary step prior to hydrolysis. Cellobiohydrolase appears to be the key enzyme for the degradation of native



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cellulose (63). The catalytic site of the enzyme is covered by long loops, resulting in tunnel morphology (64). The loops can undergo large movements, leading to the opening or closing of the tunnel roof (65). An endo type attack of the polymeric substrates becomes possible when the roof is open and once entrapped inside the catalytic tunnel, a cellulose chain is threaded through the tunnel and sequentially hydrolyzed one cellobiosyl unit at a time. Kleywegt et al. ( 66) revealed the presence of shorter loops that create a groove rather than a tunnel in the structure of the enzyme EGI from T. reesei. In most organisms, cellulases are modular enzymes that consist of a catalytic core connected to a cellulosebinding domain (CBD) through aflexibleand heavily glycosylated linker region (67). The CBD is responsible for bringing the catalytic domain in an appropriate position for the breakdown of cellulose. Binding of cellulases and the formation of cellulose-cellulase complexes are considered critical steps in the hydrolysis of insoluble cellulose (68). B-Glucosidase hydrolyzes cellobiose and in some cases cellooligosaccharides to glucose. The enzyme is generally responsible for the regulation of the whole cellulolytic process and is a rate limiting factor during enzymatic hydrolysis of cellulose as both endoglucanase and cellobiohydrolase activities are often inhibited by cellobiose (69-71). Thus, β-glueosidase not only produces glucose from cellobiose but also reduces cellobiose inhibition, allowing the cellulolytic enzymes to function more efficiently. However, like Bglucanases, most B-glucosidases are subject to end-product (glucose) inhibition (72). C. pe/tataproduces a highly glucose tolerant β-glucosidase with a value of 1.4 M (252 mg/ml) for glucose ( 73). The kinetics of the enzymatic hydrolysis of cellulose including adsorption, inactivation and inhibition of enzymes have been studied extensively (74). For a complete hydrolysis of cellulose to glucose, the enzyme system must contain the three enzymes in right proportions. T. reesei (initially called T. viride) produces at least five endoglucanases (EGI, EGII, EGIII, EGIV and EGV), two exoglucanases (CBHI and CBHII) and two βglucosidases (BGLI and BGLII) (75). An exo-exo synergism between the two cellobiohydrolases was also observed (76). The fungus produces up to 0.33 g protein per g of utilizable carbohydrate (77). Product inhibition, thermal inactivation, substrate inhibition, low product yield and high cost of cellulase are some barriers to commercial development of the enzymatic hydrolysis of cellulose. Many microorganisms are cellulolytic. However, only two microorganisms (Trichoderma and Aspergillus) have been studied extensively for cellulase. A newly isolated Mucor circinelloides strain produces a complete cellulase enzyme system (78). The endoglucanase from this strain was found to have a wide pH stability and activity. There is an increasing demand for the development of thermostable, environmentally compatible, product and substrate tolerant cellulases with increased specificity and activity for application in the conversion of cellulose to glucose in the fuel ethanol industry. Thermostable cellulases offer certain advantages such as higher reaction rate,



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increased product formation, less microbial contamination, longer shelf-life, easier purification and better yield. The cellulose hydrolysis step is a significant component of the total production cost of ethanol from wood (79). Achieving a high glucose yield is necessary (>85% theoretical) at high substrate loading (>10% w/v) over short residence times (

Lignocellulose Biodegradation and Applications in Biotechnology

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